Hyaluronic Acid and Large Extracellular Vesicles (EVs) in Synovial Fluid and Plasma of Patients With End-Stage Arthritis: Positive Association of EVs to Joint Pain.

OBJECTIVE: Hyaluronic acid (HA) in synovial fluid (SF) contributes to boundary lubrication with altered levels in osteoarthritis (OA) and rheumatoid arthritis (RA). SF extracellular vesicles (EVs) may participate in arthritis by affecting inflammation and cartilage degradation. It remains unknown whether HA and EVs display joint-specific alterations in arthritic SFs. DESIGN: We investigated the numbers and characteristics of HA-particles and large EVs in SF from knees and shoulders of 8 OA and 8 RA patients and 8 trauma controls, and in plasma from 10 healthy controls and 11 knee OA patients. The plasma and SF HA concentrations were determined with a sandwich-type enzyme-linked sorbent assay, and EVs and HA-particles were characterized from plasma and unprocessed and centrifuged SFs with confocal microscopy. The data were compared according to diagnosis, location, and preanalytical processing. RESULTS: The main findings were: (1) OA and RA SFs can be distinguished from trauma joints based on the distinctive profiles of HA-particles and large EVs, (2) there are differences in the SF HA and EV characteristics between shoulder and knee joints that could reflect their dissimilar mobility, weight-bearing, and shock absorption properties, (3) EV counts in SF and plasma can positively associate with pain parameters independent of age and body adiposity, and (4) low-speed centrifugation causes alterations in the features of HA-particles and EVs, complicating their examination in the original state. CONCLUSIONS: Arthritis and anatomical location can affect the characteristics of HA-particles and large EVs that may have potential as biomarkers and effectors in joint degradation and pain.

Metabolomics of Synovial Fluid and Infrapatellar Fat Pad in Patients with Osteoarthritis or Rheumatoid Arthritis.

Osteoarthritis (OA) and autoimmune-driven rheumatoid arthritis (RA) are inflammatory joint diseases with complex and insufficiently understood pathogeneses. Our objective was to characterize the metabolic fingerprints of synovial fluid (SF) and its adjacent infrapatellar fat pad (IFP) obtained during the same surgical operation from OA and RA knees. Non-targeted metabolite profiling was performed for 5 non-inflammatory trauma controls, 10 primary OA (pOA) patients, and 10 seropositive RA patients with high-resolution mass spectrometry-based techniques, and metabolites were matched with known metabolite identities. Groupwise differences in metabolic features were analyzed with the univariate Welch's t-test and the multivariate linear discriminant analysis (LDA) and principal component analysis (PCA). Significant discrimination of metabolite profiles was discovered by LDA for both SF and IFP and by PCA for SF based on diagnosis. In addition to a few drug-derived substances, there were 16 and 13 identified metabolites with significant differences between the diagnoses in SF and IFP, respectively. The pathways downregulated in RA included androgen, bile acid, amino acid, and histamine metabolism, and those upregulated included biotin metabolism in pOA and purine metabolism in RA and pOA. The RA-induced downregulation of androgen and bile acid metabolism was observed for both SF and IFP. The levels of 11 lipid metabolites, mostly glycerophospholipids and fatty acid amides, were also altered by these inflammatory conditions. The identified metabolic pathways could be utilized in the future to deepen our understanding of the pathogeneses of OA and RA and to develop not only biomarkers for their early diagnosis but also therapeutic targets.

Synovial Fluid Fatty Acid Profiles Are Differently Altered by Inflammatory Joint Pathologies in the Shoulder and Knee Joints.

Anomalies of fatty acid (FA) metabolism characterize osteoarthritis (OA) and rheumatoid arthritis (RA) in the knee joint. No previous study has investigated the synovial fluid (SF) FA manifestations in these aging-related inflammatory diseases in the shoulder. The present experiment compared the FA alterations between the shoulder and knee joints in patients with end-stage OA or end-stage RA. SF samples were collected during glenohumeral or knee joint surgery from trauma controls and from OA and RA patients (n = 42). The FA composition of SF total lipids was analyzed by gas chromatography with flame ionization and mass spectrometric detection and compared across cohorts. The FA signatures of trauma controls were mostly uniform in both anatomical locations. RA shoulders were characterized by elevated percentages of 20:4n-6 and 22:6n-3 and with reduced proportions of 18:1n-9. The FA profiles of OA and RA knees were relatively uniform and displayed lower proportions of 18:2n-6, 22:6n-3 and total n-6 polyunsaturated FAs (PUFAs). The results indicate location- and disease-dependent differences in the SF FA composition. These alterations in FA profiles and their potential implications for the production of PUFA-derived lipid mediators may affect joint lubrication, synovial inflammation and pannus formation as well as cartilage and bone degradation and contribute to the pathogeneses of inflammatory joint diseases.

Characterization of hyaluronan-coated extracellular vesicles in synovial fluid of patients with osteoarthritis and rheumatoid arthritis.

BACKGROUND: Hyaluronic acid (HA) is the major extracellular matrix glycosaminoglycan with a reduced synovial fluid (SF) concentration in arthropathies. Cell-derived extracellular vesicles (EV) have also been proposed to contribute to pathogenesis in joint diseases. It has recently been shown that human SF contains HA-coated EV (HA-EV), but their concentration and function in joint pathologies remain unknown. METHODS: The aim of the present study was to develop an applicable method based on confocal laser scanning microscopy (CLSM) and image analysis for the quantification of EV, HA-particles, and HA-EV in the SF of the human knee joint. Samples were collected during total knee replacement surgery from patients with end-stage rheumatoid arthritis (RA, n = 8) and osteoarthritis (OA, n = 8), or during diagnostic/therapeutic arthroscopy unrelated to OA/RA (control, n = 7). To characterize and quantify EV, HA-particles, and HA-EV, SF was double-stained with plasma membrane and HA probes and visualized by CLSM. Comparisons between the patient groups were performed with the Kruskal-Wallis analysis of variance. RESULTS: The size distribution of EV and HA-particles was mostly similar in the study groups. Approximately 66% of EV fluorescence was co-localized with HA verifying that a significant proportion of EV carry HA. The study groups were clearly separated by the discriminant analysis based on the CLSM data. The intensities of EV and HA-particle fluorescences were lower in the RA than in the control and OA groups. CONCLUSIONS: CLSM analysis offers a useful tool to assess HA-EV in SF samples. The altered EV and HA intensities in the RA SF could have possible implications for diagnostics and therapy.

Materials and Orthopedic Applications for Bioresorbable Inductively Coupled Resonance Sensors.

Bioresorbable passive resonance sensors based on inductor-capacitor (LC) circuits provide an auspicious sensing technology for temporary battery-free implant applications due to their simplicity, wireless readout, and the ability to be eventually metabolized by the body. In this study, the fabrication and performance of various LC circuit-based sensors are investigated to provide a comprehensive view on different material options and fabrication methods. The study is divided into sections that address different sensor constituents, including bioresorbable polymer and bioactive glass substrates, dissolvable metallic conductors, and atomic layer deposited (ALD) water barrier films on polymeric substrates. The manufactured devices included a polymer-based pressure sensor that remained pressure responsive for 10 days in aqueous conditions, the first wirelessly readable bioactive glass-based resonance sensor for monitoring the complex permittivity of its surroundings, and a solenoidal coil-based compression sensor built onto a polymeric bone fixation screw. The findings together with the envisioned orthopedic applications provide a reference point for future studies related to bioresorbable passive resonance sensors.

Distinct fatty acid signatures in infrapatellar fat pad and synovial fluid of patients with osteoarthritis versus rheumatoid arthritis.

BACKGROUND: Infrapatellar fat pad (IFP) has recently emerged as a potential source of inflammation in knee arthropathies. It has been proposed to be one source of adipocytokines, fatty acids (FA), and FA-derived lipid mediators that could contribute to the pathophysiological processes in the knee joint. Alterations in synovial fluid (SF) lipid composition have been linked to both osteoarthritis (OA) and rheumatoid arthritis (RA). The aim of the present study was to compare the FA signatures in the IFP and SF of RA and OA patients. METHODS: Pairs of IFP and SF samples were collected from the same knees of RA (n = 10) and OA patients (n = 10) undergoing total joint replacement surgery. Control SF samples (n = 6) were harvested during diagnostic or therapeutic arthroscopic knee surgery unrelated to RA or OA. The FA composition in the total lipids of IFP and SF was determined by gas chromatography with flame ionization and mass spectrometric detection. RESULTS: Arthropathies resulted in a significant reduction in the SF proportions of n-6 polyunsaturated FA (PUFA), more pronouncedly in OA than in RA. OA was also characterized with reduced percentages of 22:6n-3 and lower product/precursor ratios of n-3 PUFA. The proportions of total monounsaturated FA increased in both RA and OA SF. Regarding IFP, RA patients had lower proportions of 20:4n-6, total n-6 PUFA, and 22:6n-3, as well as lower product/precursor ratios of n-3 PUFA compared to OA patients. The average chain length of SF FA decreased in both diagnoses and the double bond index in OA. CONCLUSIONS: The observed complex alterations in the FA signatures could have both contributed to but also limited the inflammatory processes and cartilage destruction in the RA and OA knees.

Effects of knee flexion and extension on the tibial tuberosity-trochlear groove (TT-TG) distance in adolescents.

BACKGROUND: Measurement of the tibial tubercle-trochlear groove (TT-TG) distance is used to assess patellofemoral instability and rotation. Since patellofemoral instability and acute patellar dislocation are common among adolescents, it is important to clarify the relationship between TT-TG distance and various flexion and extension angles in asymptomatic children. The purpose of the present study was to determine how knee flexion and extension influence TT-TG-distance values measured using 3D imaging in an anatomic axial plane among asymptomatic adolescents. METHODS: We performed magnetic resonance imaging (MRI) of 26 knees in 13 adolescents (8 boys and 5 girls) of 11-17 years of age, with no known patellofemoral disorders. Imaging was performed with 3.0 T MRI with the knee at four separate angles of flexion between 0° and 30°. Measurements were made by two independent blinded raters. RESULTS: The mean TT-TG distance in millimetres was 11.1-0.29 × the angle in degrees. TT-TG distance decreased with greater flexion, showing a mean decrease of 0.29 mm (SD, 0.04) per degree of increased flexion (p < 0.001). We found significant inter-observer (Pearson's r = 0.636, p = 0.03) and intra-observer (Pearson's r = 0.792, p ≤ 0.001) correlations. TT-TG values were not significantly correlated with age, length, weight, or body mass index. The rate of TT-TG change (change between consecutive TT-TG values/change between consecutive angles) was significantly negatively correlated with length (p = 0.014), weight (p = 0.004), and body mass index (p = 0.025). CONCLUSIONS: Our data revealed that TT-TG distance assessed in the anatomic axial plane decreased with greater flexion in adolescent. Moreover, this effect of knee angle was stronger in smaller subjects. These findings support the need for a standardized protocol for TT-TG distance measurement in adolescents.

Arthroscopic partial meniscectomy versus placebo surgery for a degenerative meniscus tear: a 2-year follow-up of the randomised controlled trial.

OBJECTIVE: To assess if arthroscopic partial meniscectomy (APM) is superior to placebo surgery in the treatment of patients with degenerative tear of the medial meniscus. METHODS: In this multicentre, randomised, participant-blinded and outcome assessor-blinded, placebo-surgery controlled trial, 146 adults, aged 35-65 years, with knee symptoms consistent with degenerative medial meniscus tear and no knee osteoarthritis were randomised to APM or placebo surgery. The primary outcome was the between-group difference in the change from baseline in the Western Ontario Meniscal Evaluation Tool (WOMET) and Lysholm knee scores and knee pain after exercise at 24 months after surgery. Secondary outcomes included the frequency of unblinding of the treatment-group allocation, participants' satisfaction, impression of change, return to normal activities, the incidence of serious adverse events and the presence of meniscal symptoms in clinical examination. Two subgroup analyses, assessing the outcome on those with mechanical symptoms and those with unstable meniscus tears, were also carried out. RESULTS: In the intention-to-treat analysis, there were no significant between-group differences in the mean changes from baseline to 24 months in WOMET score: 27.3 in the APM group as compared with 31.6 in the placebo-surgery group (between-group difference, -4.3; 95% CI, -11.3 to 2.6); Lysholm knee score: 23.1 and 26.3, respectively (-3.2; -8.9 to 2.4) or knee pain after exercise, 3.5 and 3.9, respectively (-0.4; -1.3 to 0.5). There were no statistically significant differences between the two groups in any of the secondary outcomes or within the analysed subgroups. CONCLUSIONS: In this 2-year follow-up of patients without knee osteoarthritis but with symptoms of a degenerative medial meniscus tear, the outcomes after APM were no better than those after placebo surgery. No evidence could be found to support the prevailing ideas that patients with presence of mechanical symptoms or certain meniscus tear characteristics or those who have failed initial conservative treatment are more likely to benefit from APM.

Atomic layer deposition-A novel method for the ultrathin coating of minitablets.

We introduce atomic layer deposition (ALD) as a novel method for the ultrathin coating (nanolayering) of minitablets. The effects of ALD coating on the tablet characteristics and taste masking were investigated and compared with the established coating method. Minitablets containing bitter tasting denatonium benzoate were coated by ALD using three different TiO2 nanolayer thicknesses (number of deposition cycles). The established coating of minitablets was performed in a laboratory-scale fluidized-bed apparatus using four concentration levels of aqueous Eudragit® E coating polymer. The coated minitablets were studied with respect to the surface morphology, taste masking capacity, in vitro disintegration and dissolution, mechanical properties, and uniformity of content. The ALD thin coating resulted in minimal increase in the dimensions and weight of minitablets in comparison to original tablet cores. Surprisingly, ALD coating with TiO2 nanolayers decreased the mechanical strength, and accelerated the in vitro disintegration of minitablets. Unlike previous studies, the studied levels of TiO2 nanolayers on tablets were also inadequate for effective taste masking. In summary, ALD permits a simple and rapid method for the ultrathin coating (nanolayering) of minitablets, and provides nanoscale-range TiO2 coatings on porous minitablets. More research, however, is needed to clarify its potential in tablet taste masking applications.

Surface modification of acetaminophen particles by atomic layer deposition.

Active pharmaceutical ingredients (APIs) are predominantly organic solid powders. Due to their bulk properties many APIs require processing to improve pharmaceutical formulation and manufacturing in the preparation for various drug dosage forms. Improved powder flow and protection of the APIs are often anticipated characteristics in pharmaceutical manufacturing. In this work, we have modified acetaminophen particles with atomic layer deposition (ALD) by conformal nanometer scale coatings in a one-step coating process. According to the results, ALD, utilizing common chemistries for Al2O3, TiO2 and ZnO, is shown to be a promising coating method for solid pharmaceutical powders. Acetaminophen does not undergo degradation during the ALD coating process and maintains its stable polymorphic structure. Acetaminophen with nanometer scale ALD coatings shows slowed drug release. ALD TiO2 coated acetaminophen particles show cytocompatibility whereas those coated with thicker ZnO coatings exhibit the most cytotoxicity among the ALD materials under study when assessed in vitro by their effect on intestinal Caco-2 cells.

First in vivo detection and characterization of hyaluronan-coated extracellular vesicles in human synovial fluid.

Extracellular vesicles (EVs) function in intercellular signaling by transporting different membrane and cytosolic molecules, including hyaluronan (HA) and its synthesis machinery. As both EVs and HA are abundant in synovial fluid, we hypothesized that HA synthesized in synovial membrane would be carried on the surface of EVs. Synovial fluid (n = 15) and membrane samples (n = 5) were obtained from knee surgery patients. HA concentrations were analyzed in synovial fluid and HA and its synthesis machinery were examined with histochemical stainings in synovial membrane. To assess the size distribution of EVs in synovial fluid and to visualize HA on EVs, nanoparticle tracking analysis (NTA), confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM) were utilized. The average HA concentration in synovial fluid was 2.0 ± 0.21 mg/ml without significant differences between the patients with trauma/diagnostic arthroscopy and primary or post-traumatic osteoarthritis. Positive stainings of HA synthases (HAS1-3), HA and its receptor CD44 in synovial cells indicated active HA secretion in synovial membrane. According to NTA, EVs were abundant in synovial fluid and their main populations were ≤300 nm in diameter after differential centrifugation. There were no significant differences in the EV counts between the patients with primary or post-traumatic osteoarthritis. TEM verified that HA-positive particles detected by CLSM were lipid membrane vesicles surrounded by a HA coat. Our results provide the first in vivo evidence that human synovial fluid contains HA-positive EVs, one source of which presumably is the long HAS-positive protrusions of synovial fibroblasts. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1960-1968, 2016.

The effect of decompressive surgery on lumbar paraspinal and biceps brachii muscle function and movement perception in lumbar spinal stenosis: a 2-year follow-up.

PURPOSE: Chronic low back pain and lumbar spinal stenosis (LSS) seem to deteriorate lumbar muscle function and proprioception but the effect of surgery on them remains unclear. This study evaluates the effect of decompressive surgery on lumbar movement perception and paraspinal and biceps brachii (BB) muscle responses during sudden upper limb loading in LSS. METHODS: Low back and radicular pain intensity (VAS) and Oswestry Disability Index (ODI) were measured together with lumbar proprioception and paraspinal and BB muscle responses prior to and 3 and 24 months after surgery in 30 LSS patients. Lumbar proprioception was assessed by a previously validated motorized trunk rotation unit and muscle responses for sudden upper limb loading by surface EMG. RESULTS: Lumbar perception threshold improved after surgery during 3-month follow-up (from 4.6° to 3.1°, P = 0.015) but tend to deteriorate again during 24 months (4.0°, P = 0.227). Preparatory paraspinal and BB muscle responses prior to sudden load as well as paraspinal muscle activation latencies after the load remained unchanged. CONCLUSION: Impaired lumbar proprioception seems to improve shortly after decompressive surgery but tends to deteriorate again with longer follow-up despite the sustaining favorable clinical outcome. The surgery did not affect either the feed-forward or the feed-back muscle function, which indicates that the abnormal muscle activity in LSS is at least partly irreversible.

Lumbar paraspinal and biceps brachii muscle function and movement perception in lumbar spinal stenosis.

INTRODUCTION: Impaired muscle function and lumbar proprioception have been observed in lumbar spinal stenosis (LSS) but those have not been studied in LSS patients with age-matched controls. We assessed lumbar movement perception and paraspinal and biceps brachii (BB) muscle responses during sudden upper limb loading in age-matched healthy subjects and patients with LSS. METHODS: The study included 30 patients selected for an operation due to LSS and 30 age-matched controls without chronic back pain. The paraspinal and BB muscle responses for upper limb loading during unexpected and expected conditions were measured by surface EMG. The ability to sense lumbar rotation was assessed in a previously validated motorized trunk rotation unit in a seated position. Pain, disability and depression scores were recorded. RESULTS: Patients had poorer lumbar perception (mean difference 2.3 ± 0.6°, P < 0.001) and longer paraspinal muscle response latencies [mean difference 4.6 ± 0.6 ms (P = 0.033)] than age-matched healthy controls. Anticipation increased paraspinal and BB muscle activation prior to the load perturbation (P < 0.001) but less in LSS patients than in controls [9 vs. 30 %, P = 0.016 (paraspinals); 68 vs. 118 %, P = 0.047 (BB)]. CONCLUSIONS: The observed impairments in lumbar proprioception and activation of paraspinal and upper limb muscles indicate an extensive loss of both sensory and motor functions in LSS. The main new finding was decreased anticipatory muscle activation during expected upper limb loading reflecting involvement of central movement control mechanisms.